Simultaneous determination of cytokinins by high performance liquid chromatography with resonance Rayleigh scattering and mechanism discussion.

A reliable, highly sensitive and highly selective method of high performance liquid chromatography associated with resonance Rayleigh scattering (HPLC-RRS) was developed to detect three cytokinins, namely, 6-benzylaminopurine (BA), kinetin (KT) and zeatin (ZT). In this work, Pd(ii) is added into the system to form ternary ion association complexes for the first time, which results in a lower limit of detection and extends the application of HPLC-RRS. The experimental conditions were optimized. In order to investigate the reaction mechanism, the ternary ion association complexes were characterized by ultraviolet-visible spectrophotometry, dynamic light scattering, scanning electron microscopy and density functional theory calculations. In a HAc-NaAc buffer solution (pH = 4.1), a ternary complex of cytokinin : Pd(ii) : EryB (1 : 1 : 2) was formed. The detection limits (S/N = 3) of BA, KT, and ZT were 0.9, 1.5 and 2.3 ng mL-1, respectively. In addition, this method was applied for the simultaneous detection of cytokinins in real samples with satisfactory results.

Cauliflower-like resin microspheres with tuneable surface roughness as solid-phase extraction adsorbent for efficient extraction and determination of plant growth regulators in cucumbers.

New cauliflower-like phloroglucinol-glyoxylic acid resin microspheres (PGRMs) with controllable diameters and tuneable surface roughness were prepared using a one-step environmentally-friendly method without a catalyst. The PGRMs obtained exhibited a rough surface, narrow size distribution, and excellent adsorption capacity for polar compounds. The PGRMs were employed as an adsorbent for solid phase extraction (SPE) of kinetin (KT) and 6-benzyladenine (6-BA) in cucumbers and demonstrated better extraction recoveries and purification efficiency than phloroglucin-formaldehyde resin and common commercial adsorbents. Our PGRMs-SPE-HPLC method showed good linearity (r ≥ 0.9997) ranging from 0.04 to 4.00 µg/g for KT and 6-BA, and recoveries at three spiked concentration ranged from 77.8% to 104.4% with RSDs ≤ 6.8%. This PGRMs-SPE-HPLC method was applied successfully to determine of KT and 6-BA in cucumbers.

Polyhedral oligomeric silsesquioxane grafted silica-based core-shell microspheres for reversed-phase high-performance liquid chromatography.

Polyhedral oligomeric silsesquioxane (POSS) was used to modify spherical silica to fabricate core-shell POSS@SiO2 microspheres. The material was characterized by Fourier transform infrared experiments, scanning electron microscopy, thermogravimetric analysis and elemental analysis. The material was also used as a stationary phase for HPLC separation. The POSS@SiO2 column exhibits a reverse-phase liquid chromatography (RPLC) retention mechanism. The column efficiency of alkylbenzenes reaches 67,200 plates·m-1. The POSS@SiO2 column was also utilized for separation of basic anilines and polycyclic aromatic hydrocarbons. Compared with the commercial C8 column, the POSS@SiO2 column exhibits enhanced separation selectivity. The column was also used for the separation of synthetic cytokinins 6-benzylaminopurine and 6-furfurylaminopurine in bean sprout after extraction. In addition, the methacrylate groups on the surface of the POSS@SiO2 microsphere were further functionalized so as to facilitate the fabrication of versatile stationary phases with various separation mechanisms. Graphical abstract Schematic presentation of the two-step fabrication of polyhedral oligomeric silsesquioxane grafted silica-based (POSS@SiO2) core-shell microspheres for use in HPLC.

Different forms of copper and kinetin impacted element accumulation and macromolecule contents in kidney bean (Phaseolus vulgaris) seeds.

The relationship between engineered nanomaterials and plant biostimulants is unclear. In this study, kidney bean (Phaseolus vulgaris) plants were grown to maturity (90 days) in soil amended with nano copper (nCu), bulk copper (bCu), or copper chloride (CuCl2) at 0, 50, or 100 mg kg-1, then watered with 0, 10, or 100 µM of kinetin (KN). Seeds were harvested and analyzed via ICP-OES and biochemical assays. While seed production was largely unaffected, nutritional value was significantly impacted. Accumulation of Cu was enhanced by 5-10% from controls by Cu-based treatments. Fe was the only macro/microelement significantly altered by nCu, which was ~29% lower than seeds from untreated plants. All forms of Cu combined with 10 µM KN reduced Mg from 9 to 12%. Application of KN plus bCu or CuCl2 elevated concentrations of Mn (31-41%) and S (19-22%), respectively. Protein content of seeds was stimulated (11-12%) by bCu, on average, and depressed by CuCl2 + KN (up to 22%). Variations in sugar and starch content were insignificant, compared to controls. Our results indicate that the interaction Cu × KN significantly altered the nutritional value of common beans, which has potential implications to agricultural practices incorporating Cu as either a pesticide or fertilizer.

[Determination of 6 kinds of plant growth regulator in bean sprout by ultra high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: A method for the simultaneous determination of 6 plant growth regulator (PGR) residues in bean sprout was developed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 6-Benzylaminopurine, isopentennyladenine, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, indole-3- acetic acid and indole-3-butyric acid were concerned. METHODS: Bean sprout samples were extracted by acetonitrile and QuEChERS extraction kit, purified by C18 powers. After centrifugation, the sample liquids was diluted 10 times by ultrapure water. The chromatographic analysis was carried out on an waters acquity UPLC BEH C18 column( 100 mm x 2.1 mm, 1.7 microm). The analyzer confirmed and quantified by mass spectrum of triple quadrupole in the multiple reaction monitoring (MRM) mode and quantified by matrix-matched external standard method. RESULTS: The calibration curves showed good linearity in each range with correlation coefficients greater than 0.998. 3 levels spiked recoveries were carried out using blank bean sprout extraction as substrate, the recoveries ranged from 84.2% to 107.5%, the relative standard deviations (RSDs) ranged from 3.08% to 12.71%. The qualitative limits of detections (S/N = 3) were 0.03-3.0 microg/kg and the quantitative limits(S/N = 10) were 0.1-10.0 microg/kg for the 6 PGRs. CONSLUSION: The method is simple and easy to operate, with less organic reagent, high sensitivity and good stability. It is suitable for the detection of 6 kinds of plant growth regulators in bean sprouts.

[Determination of four kinds of illegal additive residues in sprouts and source beans by high performance liquid chromatography-tandem mass spectrometry].

A reversed-phase high performance liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) method was developed for the determination of 4-chlorophenoxyacetic acid, 6-benzylaminopurine, enrofloxacin and norfloxacin residues in sprouts and source beans. The sample was extracted by acetonitrile containing 0.1% acetic acid and concentrated. The chromatographic analysis was carried out on a C18 column with methanol and 0.1% formic acid solution as the mobile phases in gradient elution program. The MS analysis was set in electrospray ionization mode and separated to two segments of positive and negative modes. The precursor ions were m/z 189.9, 226.1, 359.9 and 320.1, while the product ions for quantification were m/z 127.0, 91.2, 315.9 and 276.2 for 4-chlorophenoxyacetic acid, 6-benzylaminopurine, enrofloxacin and norfloxacin, respectively. The calibration curves showed good linearity in the range of 5 - 200 microg/L with correlation coefficients more than 0.995. The limits of detection (LODs) were 1 microg/kg and the limits of quantification (LOQs) were 5 microg/kg for the four compounds spiked in mung bean sprouts and mung beans. The recoveries of the four compounds spiked at three levels of 5.0, 10.0 and 20.0 microg/kg ranged from 70% to 91%, with the relative standard deviations (RSDs) less than 14%. The method established is accurate, sensitive, simple, and has considerable advantages in the analysis of the four kinds of illegal additive residues in sprouts and beans simultaneously.

[Rapid detection of 6-benzylaminopurine residues in sprout beans by surface-enhanced raman spectroscopy].

New method for rapid and quantitative analysis of 6-benzylaminopurine (6-BA) residues in sprout beans was studied by using FAST-SPE and portable surface-enhanced Raman spectroscopy (SERS). With homemade sprout bean samples as blank control, 6-BA solutions extracted from inserted-treatment samples showed typical characteristic Raman peaks at 1002 cm(-1) tested by SERS, and normalized 1002 cm(-1) intensities had a good linear relationship with gradient concentrations of 6-BA insert-standard solutions. The high concentration linear range was 0.5-14 microg x mL(-1), and the low one was 0. 1-2 microg x mL(-1). The quantitative detection limit was down to 0.02 mg x kg(-1) that was below the maximum allowable residues (MAL) of 0. 2 mg kg(-1) in related National Standard. The recoveries were 82.3%-95.1% and the RSD was below 5%. This method with portable equipment and simple pre-treatment showed good reproducibility, short test time-consuming and easy operating, and thus it would provide efficient and sensitive solutions for large-scale on-site and rapid detections.

A small molecule screening strategy with validation on human leukemia stem cells uncovers the therapeutic efficacy of kinetin riboside.

Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34(+)CD38(-) AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias.

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method.

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA(3)), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35°C) and flow rate (1ml/min). This method presented an excellent linearity (0.2-100µg/ml) with limit of detection (LOD) as 0.2µg/ml for ABA, 0.5µg/ml for KR and salicylic acid, and 1µg/ml for IAA, IBA and GA(3). The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80-120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.

Multiresidue analysis of plant growth regulators in grapes by triple quadrupole and quadrupole-time of flight-based liquid chromatography/mass spectrometry.

A selective and sensitive LC-MS/MS method is presented for simultaneous determination of 12 plant growth regulators, viz., indol-3-acetic acid, indol-3-butyric acid, kinetin, zeatin, 6-benzyl aminopurine, gibberellic acid, abscisic acid, chlormequat chloride, forchlorfenuron, paclobutrazole, daminozide, and 2,4-dichlorophenoxy acetic acid, in bud sprouts and grape berries. The sample preparation method involved extraction of homogenized sample (5 g) with 40 mL methanol (80%), and final determination was by LC-MS/MS in the multiple reaction monitoring (MRM) mode with time segmentation for quantification supported by complementary analysis by quadrupole-time of flight (Q-TOF) MS with targeted high-resolution MS/MS scanning for confirmatory identification based on accurate mass measurements. The recovery of the test compounds ranged within 90-107% with precision RSD less than 5% (n = 6). The method could be successfully applied in analyzing incurred residue samples, and the strength of accurate mass analysis could be utilized in identifying the compounds in cases where the qualifier MRM ions were absent or at an S/N less than 3:1 due to low concentrations.

Synthesis of deuterated benzyladenine and its application as a surrogate.

Palladium on carbon-ethylenediamine complex [Pd/C(en)] catalyzed deuteration of N(6)-benzyladenine-d(5), which is a plant growth regulator, to introduce 5 deuterium atoms, while use of Pd/C as a catalyst led to a complete removal of N(6)-benzyl group. The corresponding deuterated N(6)-benzyladenine was successfully used as a surrogate compound for the quantitative analysis of residual benzyladenine in crops using LC/MS/MS.

Evaluation of matrix effects in metabolite profiling based on capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry.

The coupling of liquid chromatography to electrospray ionization quadrupole time-of-flight mass spectrometry can be a powerful tool for metabolomics, i.e., the comprehensive detection of low molecular weight compounds in biological systems. There have, however, been doubts about the feasibility and reliability of this approach, because LC-MS--especially with electrospray ionization--can be subject to matrix effects. We evaluated matrix effects for our metabolomics platform in three ways: (i) postextraction addition of a set of reference compounds to different complex biological matrixes to determine absolute and relative matrix effects, (ii) postcolumn infusion of two reference compounds, and (iii) mixing of two complex matrixes. Our data demonstrate that there are indeed significant absolute matrix effects when comparing highly divergent samples. However, relative matrix effects are negligible--unless extremely divergent matrixes are compared--and do not compromise the relative quantification that is aimed for in nontargeted metabolomics studies. In conclusion, employing LC-coupled ESI-QTOF-MS for metabolomics studies is feasible yet rigorous validation is necessary.

Electrochemical behavior and detection of plant hormone 6-benzyl adenine in acetate buffer at mercury electrode.

The electrochemical behavior of 6-benzyl adenine (6-BA) has been studied in 0.1 mol L(-1) HAc-NaAc solution (pH 3.8). Cyclic voltammetry, single-sweep polarography and direct current polarography were employed to clarify the mechanism of the electrode process; the kinetic parameters of the rate-determining step were determined. Reduction of 6-BA involves two pH-dependent processes, corresponding to the overall irreversible reduction of the 1,6 and 3,2 N=C bonds. Each reduction stage consists a preprotonation of the nitrogen atom at the electroactive site and a rapid two-electron transfer. In the presence of 6-BA, the reduction potentials of some ions were shifted. Under the given conditions, 6-BA displays one irreversible reduction peak controlled by adsorption. Two linear response were observed in the range 2.0 x 10(-8) - 8.0 x 10(-7) mol L(-1) and the range 1.0 x 10(-6) - 8.0 x 10(-6) mol L(-1), with correlation coefficients of 0.9995 and 0.9998, respectively. The detection limit is 7.1 x 10(-9) mol L(-1). The method had been applied to the determination of 6-BA in bean sprout samples with satisfactory results.

Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography-tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis.

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits ("coconut water"). To facilitate the study, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC-MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 microM and 0.005 microM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures.

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.